IFN-γ诱导的自噬对奶牛乳腺上皮细胞泌乳与细胞增殖及抗细菌感染的调控

发布时间：2018-06-04 14:08

本文选题：IFN-γ + 奶牛乳腺上皮细胞　；参考：《吉林大学》2016年博士论文

【摘要】：营养直接影响机体的免疫状况,越来越多的研究证实日粮可以改变炎性细胞因子的释放,为一些病症的病因探讨提供了新方向。我国优质牧草资源缺乏,秸秆粗饲料配合高精料的日粮模式很普遍。课题组在前期研究发现,长期饲喂这种秸杆日粮的奶牛IFN-γ等细胞因子的分泌水平较高,可能会导致机体出现非感染性炎症状态,成为多种疾病的诱因,但是,IFN-γ对奶牛乳腺疾病的影响尚不清楚。为此,本研究在建立原代奶牛乳腺上皮细胞(Bovine mammary epithelial cells,BMECs)体外培养平台的基础上,开展了IFN-γ对细胞增殖、乳脂乳蛋白合成及对病原的易感性等方面的影响及其分子机制研究;并对奶牛乳腺进行活体采样,进行了体内验证。为饲料营养通过免疫调控乳腺泌乳及抗病力提供了新的基础。通过活检技术获得奶牛乳腺组织,应用免疫组化等技术检测显示秸秆饲料组(Corn straw,CS)较苜蓿组(Mixed forage,MF)IFN-γ及其受体(IFNGR1、IFNGR2)表达显著上调;自噬分子标志物LC3-II及P62表达显著变化,说明秸秆组自噬增强。为明确IFN-γ能否诱导BMECs自噬,运用Western blotting及透射电镜分析等实验方法分析IFN-γ诱导BMECs发生自噬。研究结果显示IFN-γ处理BMECs,其自噬标志蛋白LC3-II表达升高,LC3点状聚集物的形成以及自噬溶酶体等显微结构增多,自噬底物p62的降解增加。表明IFN-γ体内体外均可诱导BMECs自噬。为进一步探索IFN-γ诱导BMECs自噬的分子机制,本研究应用质谱、Western blotting等实验技术证实IFN-γ加速精氨酸耗竭,激活氨基酸感受器GCN2,增加e IF2a磷酸化调控自噬。相反,补充精氨酸可减少GCN2/e IF2a信号通路活化并抑制自噬。进一步通过RNA干扰GCN2明显抑制了IFN-γ诱导的BMECs中LC3-II的表达及p62的降解。表明IFN-γ加速精氨酸耗竭活化GCN2/e IF2α信号途径介导了BMECs细胞自噬。为确定自噬的发生对BMECs功能的影响,应用MTT、流式细胞术等方法检测显示激活自噬加速BMECs细胞周期进程,细胞生长加快,在软琼脂中形成细胞克隆能力增强表明BMECs发生恶性转化。进一步研究显示,上皮细胞-间充质细胞转化(EMT)并不参与这一过程,而自噬促进c-Abl入核增加HDAC2的稳定性,加强对细胞周期相关蛋白的调控,加速细胞周期进程。泌乳是BMECs最重要的功能,本研究发现IFN-γ通过活化自噬抑制乳脂乳蛋白的合成。通过自噬抑制剂干预自噬潮的形成,能够逆转IFN-γ抑制的乳脂乳蛋白合成,而自噬激动剂诱导自噬潮的形成,能够加剧IFN-γ抑制的乳脂乳蛋白合成。提示自噬介导了IFN-γ抑制的乳脂乳蛋白合成。补充精氨酸可减少GCN2/e IF2a信号通路活化并抑制自噬恢复乳脂肪乳蛋白合成。应用电镜观察等方法探讨了IFN-γ诱导的自噬在BMECs抵抗金黄色葡萄球菌感染中的作用。结果显示ITGA4参与了金黄色葡萄球菌粘附和侵入BMECs。透射电镜观察发现双层膜结构包裹菌体,平板计数显示抑制自噬,胞内菌明显增加,相反,增强自噬,胞内菌计数明显减少。表明自噬促进胞内菌清除。综上所述,IFN-γ在体内体外促进自噬。精氨酸耗竭活化GCN2/e IF2a信号途径介导了自噬的发生。自噬促进细胞恶性增殖、转化,抑制乳脂乳蛋白合成,有助于BMECs胞内金黄色葡萄球菌清除。本研究揭示了IFN-γ在BMECs诱导自噬及其分子机制,不仅为乳脂乳蛋白合成调控与乳腺免疫防御开辟了新的领域,也为防治乳腺相关疾病提供了新的思路与实验线索。
[Abstract]:Nutrition directly affects the immune status of the body. More and more studies have confirmed that diet can change the release of inflammatory cytokines and provide a new direction for the discussion of the etiology of some diseases. The lack of high quality pasture resources in China is very common. The secretory level of IFN- gamma and other cytokines in dairy cows on straw diet is high, which may lead to non infectious inflammatory state and become a cause of various diseases. However, the effect of IFN- gamma on mammary disease of dairy cows is not clear. Therefore, this study is to establish the primary mammary epithelial cells (Bovine mammary epithelial cells, BMECs). On the basis of the external culture platform, the effects of IFN- gamma on cell proliferation, lactating milk protein synthesis and susceptibility to pathogenic factors and its molecular mechanism were carried out. The expression of Corn straw (CS) in the straw feed group (Mixed forage, MF) IFN- y and its receptor (IFNGR1, IFNGR2) was significantly up-regulated in the straw feed group (Mixed forage, MF), and the expression of the autophagy marker LC3-II and P62 was significantly increased. Western blotting and transmission electron microscopy were used to analyze the autophagy of BMECs induced by IFN- gamma. The results showed that IFN- gamma treated BMECs, the expression of autophagic marker protein LC3-II increased, LC3 dot aggregation, autophagosome and other microstructures increased, and the degradation of autophagic substrate p62 increased. The results showed that IFN- gamma in vitro and in vivo were both in vitro and in vitro In order to further explore the autophagy of BMECs. In order to further explore the molecular mechanism of IFN- gamma induced autophagy, this study uses mass spectrometry, Western blotting and other experimental techniques to confirm that IFN- gamma accelerates arginine depletion, activates the amino acid receptor GCN2 and increases the IF2a phosphorylation of e to regulate autophagy. On the contrary, the addition of arginine can reduce the activation and inhibition of GCN2/e IF2a signaling pathway. Autophagy. Further interfering with GCN2 by RNA interference significantly inhibits the expression of LC3-II and degradation of p62 in BMECs induced by IFN- gamma. It is indicated that IFN- gamma accelerates the depletion of arginine to activate GCN2/e IF2 alpha signal pathway to mediate the autophagy of BMECs cells. In order to determine the effect of autophagy on BMECs function, MTT, flow cytometry, and other methods are used to detect the activation of the autophagy. By accelerating the process of BMECs cell cycle and accelerating cell growth, the enhancement of cell cloning ability in soft agar indicates a malignant transformation of BMECs. Further research shows that epithelial cell mesenchymal cell transformation (EMT) does not participate in this process, and autophagy promotes the stability of c-Abl into the nucleus and increases the stability of HDAC2, and strengthens the cell cycle related proteins. Regulation, accelerating cell cycle process. Lactating is the most important function of BMECs. This study found that IFN- gamma inhibits the synthesis of milk protein by activating autophagy. By interfering with autophagy formation by autophagy inhibitors, it can reverse the synthesis of milk protein in IFN- gamma inhibition, and autophagic agonists induce the formation of autophagic tides, which can aggravate the IFN- gamma inhibition The synthesis of milk fat milk protein suggests that autophagy mediates the synthesis of milk fat milk protein in IFN- gamma inhibition. Supplementation of arginine can reduce the activation of GCN2/e IF2a signaling pathway and inhibit autophagy to restore milk fat milk protein synthesis. The effect of autophagy induced by IFN- gamma in BMECs resistance to Staphylococcus aureus infection was investigated by means of electron microscopy. The results showed that ITGA4 was involved in the adhesion and invasion of Staphylococcus aureus by BMECs. transmission electron microscopy. The double layer membrane structure was found. The plate count showed that the autophagy was inhibited and the intracellular bacteria increased obviously. On the contrary, the autophagy was enhanced and the count of intracellular bacteria decreased obviously. The autophagy promoted the removal of intracellular bacteria. In conclusion, IFN- gamma promoted autophagy in body and body in vitro and in vitro. The depletion of ammonia and activation of GCN2/e IF2a signal pathway mediates the occurrence of autophagy. Autophagy promotes cell proliferation, transformation, inhibits milk protein synthesis and helps to remove Staphylococcus aureus in BMECs cells. This study revealed that IFN- gamma induced autophagy in BMECs and its molecular mechanism, not only for milk fat milk protein synthesis regulation and mammary immune defense. It has opened up new fields and provided new clues and experimental clues for the prevention and treatment of breast related diseases.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823

【相似文献】